The effect of matrix metalloproteinase-9 on the differentiation into osteoclast cells on RAW264 cells

Matrix metalloproteinases (MMPs) break down the extracellular matrix (ECM) and are important proteins for bone remodeling. Here, to clarify relationships between MMPs and mechanisms of osteoclast differentiation, we used MMP inhibitor, GM6001, to investigate osteoclast differentiation engage to receptor activator of nuclear factor κB ligand (RANKL)/receptor activator of nuclear factor κB (RANK) stimulation using the RAW264 osteoclast precursor cell line. Also, since differentiation of RAW264 due to RANKL/RANK stimulation is dependent on p38 mitogen-activated protein kinase (p38 MAPK), we investigated expression of MMP-9 and osteocyte differentiation of RAW264 due to RANKL stimulation using SB203580, a p38 MAPK inhibitor. After confirming that GM6001 did not significantly affect cellular proliferation of the cell line, RAW264 was cultured with GM6001 and RANKL. GM6001 suppressed RANKL stimulation-induced tartrate-resistant acid phosphatase (TRAP)-positive polynuclear osteoclasts in a dose-dependent manner, and RANKL increased expression of MMP-9 in a dose-dependent manner. SB203580 also suppressed differentiation into TRAP-positive multinucleated osteocytes in a dose-dependent manner and blocked expression of MMP-9. These findings suggest that differentiation of osteoclasts by RANKL stimulation involves expression of MMP-9, which in turn involves p38 MAPK.